Agrobacterium rhizogenes transformation and expression of rol genes in Kalanchoë

ABSTRACT

The present disclosure embraces  Kalanchoë  interspecific hybrid plants, and considers rol transformation in  Kalanchoë  species and hybrids. Disclosed herein are methodology and the like for producing rol-transformed  Kalanchoë  interspecific hybrid plants, as well as resultant rol-transformed  Kalanchoë  interspecific hybrid plants with novel phenotypes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 30, 2013 isnamed 48374-0122 SL.txt and is 44,954 bytes in size.

FIELD

The present disclosure embraces Kalanchoë interspecific hybrid plants,and considers rol transformation in Kalanchoë species and hybrids.

INTRODUCTION

Kalanchoë blossfeldiana, and its cultivars, is a horticulturallyimportant plant due to its popularity as both an indoor and outdoorplant. The genus of Kalanchoë belongs to the sedum family(Crassulaceae), and there are more than 100 different species ofKalanchoë, most of which are found in Madagascar and South Africa, and afew in Asia and South America. Kalanchoë are succulent plants,characterized by turgid leaves that enable the plants to survive droughtconditions. Consequently, Kalanchoë are useful ornamental plants becausethey can survive in less than optimal growing conditions.

Kalanchoë displays an elongated growth habit in nature, which isconsidered undesirable for the potted plant industry that favors morecompact plant architecture for space and transportation purposes. Thus,the industry treats Kalanchoë plants with chemical growth retardants toalter plant shape and size.

SUMMARY

In one aspect, there is provided a species-independent method fortransforming a Kalanchoë interspecific hybrid plant, comprising: (a)co-cultivating wild-type A. rhizogenes with a Kalanchoë interspecifichybrid plant, wherein A. rhizogenes transfers one or more rol genes intosaid plant; (b) selecting a putatively transformed root having a hairyroot phenotype; (c) growing the root on a regeneration medium; (d)regenerating a shoot from the root, thereby generating a plantlet, and;(e) growing the plantlet into a mature plant. In one embodiment, themethod further comprises assaying the presence of one or more rol genesin the mature plant.

In another aspect, provided is a method for producing a Kalanchoëinterspecific hybrid plant having intermediate compactness, comprising:(a) transforming Kalanchoë plant tissue with A. rhizogenes, wherein A.rhizogenes delivers and integrates one or more rol genes into plantgenome; (b) selecting a putatively transformed root having a hairy rootphenotype; (c) growing the root on a regeneration medium; (d)regenerating a shoot from the root, thereby generating a plantlet; (e)growing said plantlet into a mature plant, and; (f) selecting a planthaving intermediate compactness, wherein intermediate compactness isfrom about 5% to about 50% of a non-transformed control plant.

In another aspect, provided is a method for reducing the height of aKalanchoë interspecific hybrid plant by about 5% to about 60%, comparedto a wild-type control plant, comprising: (a) transforming Kalanchoëplant tissue with A. rhizogenes, wherein A. rhizogenes delivers andintegrates one or more rol genes into hybrid plant genome; (b) selectinga putatively transformed root having a hairy root phenotype; (c) growingsaid root on a regeneration medium; (d) regenerating a shoot from saidroot, thereby generating a plantlet; (e) growing said plantlet into amature plant, and; (f) selecting a plant having reduced height comparedto a non-transformed control plant.

In another aspect, the disclosure provides a rol-transformed Kalanchoëinterspecific hybrid with intermediate height, wherein said intermediateheight is about 5% to about 60% of a control, non-transformed Kalanchoëinterspecific hybrid plant.

In another aspect, provided herein is a Kalanchoë interspecific hybridcomprising one or more rol genes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates double-type Kalanchoë interspecific hybrid 2006-0199plants in early generative stage after 6 weeks under short dayconditions. Plants labeled from left to right: on the left is a roltransformed line (line 6a); in the middle is an intermediate line (line3a), and on the right is an untransformed control plant.

FIG. 2 illustrates double-type Kalanchoë interspecific hybrid 2006-0199plants in generative stage after 12 weeks under short day conditions.Plants labeled from left to right: on the left is a rol transformed line(line 6a); in left middle position is an intermediate line (line 3a1),in the right middle position is an intermediate line (line 3a2) and onthe right is an untransformed control plant.

FIG. 3 depicts an illustrative A. rhizogenes R1-plasmid from an agropinestrain. The T-DNA contains two segments, T_(L) and T_(R), which areseparated by a 15 Kb sequence that is not integrated. The T_(L)-DNAcontains 18 open reading frames (ORFs) where the four root loci-genesreside. The T_(R)-DNA contains several genes, including aux1 and aux2.

DETAILED DESCRIPTION

The present disclosure embodies methodology and means for transformingKalanchoë species and hybrids with Agrobacterium rhizogenes (A.rhizogenes), as well as employing A. rhizogenes for altering Kalanchoëgrowth and plant architecture.

The Ri-plasmid of naturally occurring soil bacterium A. rhizogenesagropine-type strains carry two T-DNA regions (T_(L)-DNA and T_(R)-DNA)on the Ri-plasmid for transfer into plant cells. Following infection ofa plant cell, the bacterium transfers the entire T-DNA region (bothT_(L)-DNA and T_(R)-DNA), thereby transferring rol (root loci) genesinto the plant genome and causing hairy root growth at the site ofinfection. Tepfer (1984) Cell, 37, pp. 959-967. Because A. rhizogenesnaturally infects plants, the rol genes are naturally transferred intothe plant and function as plant oncogenes and develop hairy roots inplant tissues.

The T_(L)-DNA contains four rol genes, rolA, rolB, rolC, and rolD,whereas the T_(R)-DNA contains several genes, including two auxin genes,aux1 and aux2.

Here, the present inventors provide species-independent methodology fortransforming a Kalanchoë interspecific hybrid with A. rhizogenes, aswell as methodology for altering Kalanchoë interspecific hybrid growthand plant architecture. As described below, the present inventorsdiscovered novel phenotypes, such as intermediate plant height andcompactness, that can be obtained through rol introduction.

While any methodology can be used for producing rol-expressing Kalanchoëinterspecific hybrids, the present disclosure provides both “natural”and “non-natural” methodology for generating rol-transformed Kalanchoëinterspecific hybrids. For example, and as discussed below, Applicantsharnessed wild-type A. rhizogenes to transfer its native rol genes intoa plant cell. While this is a “natural” system in that A. rhizogenestransfers its native rol genes to plant cells, it is extremely unlikelyto occur in nature because interspecific hybrids rarely exist, let alonefertile interspecific hybrids. That is, geographical distribution ofKalanchoë species does not favor the creation of interspecific hybrids,and in the rare instance of their existence, the interspecific hybridshave low fertility and low seed dispersal. Furthermore, compact plantslike rol-transformed Kalanchoë face obstacles such as increased risk offungal infection due to compact leaves forming closed canopy structure,as well as competitiveness from neighboring plants.

All technical terms used herein are terms commonly used in biochemistry,molecular biology and agriculture, and can be understood by one ofordinary skill in the art. Technical terms can be found in: MolecularCloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook andRussell, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; Current Protocols in Molecular Biology, ed. Ausubel et al., GreenePublishing Associates and Wiley-Interscience, New York, 1988 (withperiodic updates); Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, 5th ed., vol. 1-2,ed. Ausubel et al., John Wiley & Sons, Inc., 2002; Genome Analysis: ALaboratory Manual, vol. 1-2, ed. Green et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1997. Methodology involvingplant biology techniques is described herein and is described in detailin treatises such as Methods in Plant Molecular Biology: A LaboratoryCourse Manual, ed. Maliga et al., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1995. Various techniques using PCR aredescribed in Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, San Diego, 1990 and in Dieffenbach andDveksler, PCR Primer: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2003. PCR-primer pairs canbe derived from known sequences by known techniques such as usingcomputer programs intended for that purpose, Primer, Version 0.5, 1991,Whitehead Institute for Biomedical Research, Cambridge, Mass. Methodsfor chemical synthesis of nucleic acids are discussed, for example, inBeaucage and Caruthers, 1981, Tetra. Letts. 22: 1859-1862, and Matteucciand Caruthers, 1981 J. Am. Chem. Soc. 103: 3185. Restriction enzymedigestions, phosphorylations, ligations and transformations were done asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed. (1989), Cold Spring Harbor Laboratory Press. All reagents andmaterials used for the growth and maintenance of bacterial cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), Invitrogen (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.), unless otherwise specified.

“Transformation” refers to any methodology for introducing a rol gene(s)into a host plant cell. Importantly, and because A. rhizogenes naturallyinfects plants, transformation includes the natural transfer ofwild-type rol genes from wild-type bacterium into a plant cell. Thus,and as used herein, transformation neither implies nor requires cloninga heterologous gene into a vector for transfer into a host plant cell.Furthermore, a host plant cell expressing a rol gene(s) may becharacterized as “transformed.” Transformation may occur by any knownmethod including, for example, natural infection, floral dip,infiltration, or particle bombardment. Transformation of a cell may bedetected by any known means, including but not limited to Northern Blot,Southern blot, PCR, and/or RT-PCR.

The term “tissue culture” refers to plant tissues propagated understerile conditions, often for producing clones of a plant. Plant tissueculture relies on the fact that many plant cells have the ability toregenerate a whole plant. Single cells, plant cells without cell walls(protoplasts), pieces of leaves, or roots can often be used to generatea new plant on culture media given the required nutrients and planthormones.

“Kalanchoë interspecific hybrid” embraces any Kalanchoë plant with aninterspecific cross in its background. That is, interspecific hybridsinclude both the first and subsequent generations of crosses between twoKalanchoë species, as well as the progeny produced from either selfingan interspecific hybrid or crossing an interspecific hybrid with aKalanchoë of the same or different species.

A. rhizogenes refers to Agrobacterium rhizogenes and its Ri-plasmid froman agropine strain. The T-DNA contains two segments, T_(L) and T_(R),which are separated by a 15 Kb sequence that is not integrated. TheT_(L)-DNA contains 18 open reading frames (ORFS) where the four rootloci-genes reside. The T_(R)-DNA contains several genes, including aux1and aux2.

“Hairy root phenotype” refers to a plant phenotype indicative of aputative transformed plant. That is, when A. rhizogenes infects a plantcell and transfer one or more rol genes, hairy root growth occurs at theinfection site. In this way, a hairy root phenotype offers a marker-freemethod for indentifying putative transformants.

“Intermediate height” refers to a quantitative reduction of plant heightrelative to a wild-type or control plant of the same species. The heightof the transformed plant can be decreased from about 5% to about 60%,preferably from 10% to about 50%, even more preferably from 15% to about50% of the height of a wild type plant.

“Intermediate compactness” refers to a quantitative reduction of plantcompactness relative to a wild-type or control plant of the samespecies. The compactness of the transformed plant can be increased fromabout 5% to about 50%, preferably from 10% to about 50%, even morepreferably from 15% to about 50% of the height of a wild type plant.

A. Nucleic Acid Sequences

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of RNA or apolypeptide. A polypeptide can be encoded by a full-length codingsequence or by any part thereof. The term “parts thereof” when used inreference to a gene refers to fragments of that gene, particularly afragment encoding at least a portion of a protein. The fragments mayrange in size from a few nucleotides to the entire gene sequence minusone nucleotide. Thus, “a nucleic acid sequence comprising at least apart of a gene” may comprise fragments of the gene or the entire gene.

“Gene” also encompasses the coding regions of a structural gene andincludes sequences located adjacent to the coding region on both the 5′and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated (or untranslated) sequences(5′ UTR). The sequences which are located 3′ or downstream of the codingregion and which are present on the mRNA are referred to as 3′non-translated (or untranslated) sequences (3′ UTR).

“Nucleic acid” as used herein refers to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids.

“Encoding” and “coding” refer to the process by which a gene, throughthe mechanisms of transcription and translation, provides information toa cell from which a series of amino acids can be assembled into aspecific amino acid sequence to produce an active enzyme. Because of thedegeneracy of the genetic code, certain base changes in DNA sequence donot change the amino acid sequence of a protein. It is thereforeunderstood that modifications in the DNA sequence encoding transcriptionfactors which do not substantially affect the functional properties ofthe protein are contemplated.

The term “expression,” as used herein, refers to the production of afunctional end-product e.g., an mRNA or a protein.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analog of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

Probe or primer refers to a short oligonucleotide sequence that could bedesigned and synthesized, or generated as a fragment of a largersequence. A probe or primer can be any length, such as 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides inlength.

Illustrative rol sequences include but are not limited to the sequencesset forth in SEQ ID NOs: 1-18, respectively, as well as nucleic acidmolecules comprised of fragments or variants of SEQ ID NO: 1-18 with oneor more bases deleted, substituted, inserted, or added, which variantcodes for a polypeptide with rol activity. For example, and in no waylimiting, the present disclosure provides SEQ ID NO: 1, as well asvarious fragments of SEQ ID NO: 1, which could include, for example,rolA-D and aux1-2. For instance, and as readily apparent to one ofordinary skill in the art, the rolA gene could represent a 700 bpportion or fragment of a larger sequence comprising rolA-D and aux1-2.

A “variant” is a nucleotide or amino acid sequence that deviates fromthe standard, or given, nucleotide or amino acid sequence of aparticular gene or protein. The terms “isoform,” “isotype,” and “analog”also refer to “variant” forms of a nucleotide or an amino acid sequence.An amino acid sequence that is altered by the addition, removal, orsubstitution of one or more amino acids, or a change in nucleotidesequence, may be considered a “variant” sequence. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. A variant may have “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Analogous minor variationsmay also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues may be substituted, inserted,or deleted may be found using computer programs well known in the artsuch as Vector NTT Suite (InforMax, Md.) software. “Variant” may alsorefer to a “shuffled gene” such as those described in Maxygen-assignedpatents.

Included in the category of “variant” sequences are sequences thathybridize to a reference rol sequence. For example, two sequenceshybridize when they form a double-stranded complex in a hybridizationsolution of 6×SSC, 0.5% SDS, 5×Denhardt's solution and 100 μg ofnon-specific carrier DNA. See Ausubel et al., supra, at section 2.9,supplement 27 (1994). Sequences may hybridize at “moderate stringency,”which is defined as a temperature of 60.degree. C. in a hybridizationsolution of 6.times.SSC, 0.5% SDS, 5.times. Denhardt's solution and100.mu.g of non-specific carrier DNA. For “high stringency”hybridization, the temperature is increased to 68.degree. C. Followingthe moderate stringency hybridization reaction, the nucleotides arewashed in a solution of 2.times.SSC plus 0.05% SDS for five times atroom temperature, with subsequent washes with 0.1.times.SSC plus 0.1%SDS at 60.degree. C. for 1 hour. For high stringency, the washtemperature is increased to 68.degree. C. One with ordinary skill in theart can readily select such conditions by varying the temperature duringthe hybridization reaction and washing process, the salt concentrationduring the hybridization reaction and washing process, and so forth. Forpresent purposes, hybridized nucleotides can be detected using 1 ng of aradiolabeled probe having a specific radioactivity of 10,000 cpm/ng,where the hybridized nucleotides are clearly visible following exposureto X-ray film at −70.degree. C. for no more than 72 hours.

The present application is directed to such nucleic acid molecules thatare at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a nucleic acid sequence described in any of SEQ IDNO: 1-18. Preferred are nucleic acid molecules which are at least 95%,96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shownin any of SEQ ID NO: 1-18. Differences between two nucleic acidsequences may occur at the 5′ or 3′ terminal positions of the referencenucleotide

As a practical matter, stating whether any particular nucleic acidmolecule is at least 95%, 96%, 97%, 98% or 99% identical to a referencenucleotide sequence implicates a comparison made between two molecules,using algorithms known in the art and can be determined conventionallyusing publicly available computer programs such as the blastn algorithm(National Center for Biotechnology, Bethesda, Md., US). See Altschul etal., Nucleic Acids Res. 25: 3389-402 (1997). The terms “sequenceidentity” and “sequence similarity” can be determined by alignment oftwo peptide or two nucleotide sequences using global or local alignmentalgorithms. Sequences may then be referred to as “substantiallyidentical” or “essentially similar” when they share at least 70% ofsequence identity over their entire length, respectively. Sequencealignments and scores for percentage sequence identity may be determinedusing computer programs, such as the GCG Wisconsin Package, Version10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego,Calif. 92121-3752 USA, or EmbossWin version 2.10.0 (using the program“needle”). Alternatively percent similarity or identity may bedetermined by searching against databases, using algorithm as FASTA,BLAST, etc.

The present disclosure may contemplate nucleic acid molecules encodingfunctional proteins. As known in the art, it is understood that suchproteins encompass amino acid substitutions, additions, and deletionsthat do not alter the function of any of the proteins.

Because many proteins are encoded by gene families, it is expected thatother genes could encode proteins with similar functions as the instantpolypeptides. These genes can be identified and functionally annotatedby sequence comparison. A worker skilled in the art can identify afunctionally related protein sequence with the aid of conventionalmethods such as screening cDNA libraries or genomic libraries withsuitable hybridization probes. The skilled artisan knows that paralogoussequences can also be isolated with the aid of (degenerate)oligonucleotides and PCR-based methods.

B. Nucleic Acid Constructs

As explained above, one or more rol sequences are transferred into ahost plant cell. Such transfer can occur through natural means, such asnatural infection of plant cell with A. rhizogenes carrying native rolgenes. Such natural or native transfer avoids the need for constructsand selection markers.

However, in another aspect, one or more rol sequences can beincorporated into a nucleic acid construct that is suitable forintroduction into a plant cell. Thus, in instance where a native systemis not employed, a nucleic acid construct can be used to express rol ina plant cell.

Exemplary nucleic acid constructs may comprise a base sequence of aminimum length to generate a mRNA and consequently a polypeptide. Thereis no theoretical upper limit to the base sequence length. Thepreparation of such constructs is described in more detail below.

As a source of the nucleic acid sequence for transcription, a suitablecDNA or genomic DNA or synthetic polynucleotide may be used. Methods forthe isolation of suitable rol sequences are described, supra. Sequencescoding for the whole, or substantially the whole, of the sequence maythus be obtained. Suitable lengths of this DNA sequence may be cut outfor use by means of restriction enzymes. When using genomic DNA as thesource of a partial base sequence for transcription, it is possible touse either intron or exon regions or a combination of both.

Recombinant nucleic acid constructs may be made using standardtechniques. For example, the nucleic acid sequence for transcription maybe obtained by treating a vector containing said sequence withrestriction enzymes to cut out the appropriate segment. The nucleic acidsequence for transcription may also be generated by annealing andligating synthetic oligonucleotides or by using syntheticoligonucleotides in a polymerase chain reaction (PCR) to give suitablerestriction sites at each end. The nucleic acid sequence then is clonedinto a vector containing suitable regulatory elements, such as upstreampromoter and downstream terminator sequences.

Another aspect concerns a nucleic acid construct wherein a rol sequenceis operably linked to one or more regulatory sequences, which driveexpression of the rol sequence in certain cell types, organs, or tissueswithout unduly affecting normal development or plant physiology.

Of course, and in the context of a natural transformation or naturalinfection system, native or endogenous regulatory sequences are used,rather than heterologous sequences.

“Promoter” connotes a region of DNA upstream from the start oftranscription that is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “constitutivepromoter” is one that is active throughout the life of the plant andunder most environmental conditions. Tissue-specific, tissue-preferred,cell type-specific, and inducible promoters constitute the class of“non-constitutive promoters.” “Operably linked” refers to a functionallinkage between a promoter and a second sequence, where the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. In general, “operably linked”means that the nucleic acid sequences being linked are contiguous.

Promoters useful for expression of a nucleic acid sequence introducedinto a cell may include native or endogenous promoters for naturaltransformation systems, or constitutive promoters, such as thecauliflower mosaic virus (CaMV) 35S promoter, or tissue-specific,tissue-preferred, cell type-specific, and inducible promoters. Forexample, by using vascular system-specific, xylem-specific, orxylem-preferred promoters, one can modify rol expression specifically inmany tissues such as vascular tissues, especially xylem. The use of aconstitutive promoter in general affects enzyme levels and functions inall parts of the plant, while use of a tissue-preferred promoter permitstargeting of the modified gene expression to specific plant parts,leading to a more controllable phenotypes.

A vector may also contain a termination sequence, positioned downstreamof a rol sequence, such that transcription of mRNA is terminated, andpolyA sequences added. Exemplary of such terminators are native orendogenous terminator sequenes, cauliflower mosaic virus (CaMV) 35Sterminator, or the nopaline synthase gene (Tnos) terminator. Theexpression vector also may contain enhancers, start codons, splicingsignal sequences, and targeting sequences.

Expression vectors may also contain a selection marker by whichtransformed cells can be identified in culture. The marker may beassociated with the heterologous nucleic acid molecule, i.e., the geneoperably linked to a promoter. As used herein, the term “marker” refersto a gene encoding a trait or a phenotype that permits the selection ofor the screening for, a plant or cell containing the marker. In plants,for example, the marker gene will encode antibiotic or herbicideresistance. This allows for selection of transformed cells from amongcells that are not transformed or transfected.

Examples of suitable selectable markers include adenosine deaminase,dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidnekinase, xanthine-guanine phospho-ribosyltransferase, glyphosate andglufosinate resistance, and amino-glycoside 3′-O-phosphotranserase(kanamycin, neomycin and G418 resistance). These markers may includeresistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin.The construct may also contain the selectable marker gene Bar thatconfers resistance to herbicidal phosphinothricin analogs like ammoniumgluphosinate. Thompson et al., EMBO J. 9: 2519-23 (1987). Other suitableselection markers are known as well.

Visible markers such as green florescent protein (GFP) may be used.Methods for identifying or selecting transformed plants based on thecontrol of cell division have also been described. See WO 2000/052168and WO 2001/059086. Likewise, the presence of a distinguishingphenotype, such as tumor or hairy root growth, may also be used foridentification and selection.

In a natural transformation or natural infection system, a selectionmarker is not employed. Because infection provides its own distinct andnatural phenotype, a transformed cell can be selected based on apost-infection phenotype, such as hairy root phenotype.

Replication sequences, of bacterial or viral origin, may also beincluded to allow the vector to be cloned in a bacterial or phage host.Preferably, a broad host range prokaryotic origin of replication isused. A selectable marker for bacteria may be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as kanamycin or tetracycline.

Other nucleic acid sequences encoding additional functions may also bepresent in the vector, as is known in the art. For instance, whenAgrobacterium is the host, T-DNA sequences may be included to facilitatethe subsequent transfer to and incorporation into plant chromosomes.

C. Kalanchoë Species and Interspecific Hybrids

As used herein, “interspecific hybrid” includes the progeny from thecross of two different species of Kalanchoë and its cultivars, as wellas progeny resulting from subsequent backcrossing to one of the parents.This backcrossing to one of the parents may be conducted one or moretimes with the goal of stably combining the double-type trait withdesired characteristics.

K. blossfeldiana can be crossed with numerous other Kalanchoë species tocombine advantageous characteristics into unique new cultivars. Amongthe numerous interspecific hybrids that may be created are K.blossfeldiana×K. laciniata, K. blossfeldiana×K. rotundifolia, K.blossfeldiana×K. aromatica, K. blossfeldiana×K. pubescens, K.blossfeldiana×K. grandiflora, K. blossfeldiana×K. citrina, K.blossfeldiana×K. ambolensis, K. blossfeldiana×K. faustii, Kblossfeldiana×K. schumacherii, K. blossfeldiana×K. pritwitzii, K.blossfeldiana×K. flammea, K. blossfeldiana×K. figueredoi, K.blossfeldiana×K. rauhii, K. blossfeldiana×K. obtusa, K. blossfeldiana×K.pumila, K. blossfeldiana×K. marmorata, K. blossfeldiana×K.porphyrocalux, K. blossfeldiana×K. jongmansii, K. blossfeldiana×K.pinnata, K. blossfeldiana×K. daigremontiana, K blossfeldiana×K.gracilipes, K blossfeldiana×K. campanulata, K blossfeldiana×K.latisepela, K. blossfeldiana×K. coccinea, K. blossfeldiana×K.fedtschenkoi, K. blossfeldiana×K. tubiflora, K. blossfeldiana×K.decumbens, K. blossfeldiana×K. manginii, K. blossfeldiana×K. orgyalis,K. blossfeldiana×K. crenata and K. blossfeldiana×K. tomentosa.

As a first step in making interspecific hybrids, a single or double-typeKalanchoë plant selection is crossed with a single-type Kalanchoëselection from another species. Progeny are screened for fertileselections. Large numbers of progeny may have to be screened to identifyfertile selections. The fertile selections may be screened for thoseexhibiting the double-type flower trait if one of the parents was adouble-type selection. Alternatively, the single-type fertileinterspecific hybrid is crossed, either as the male or female parent,with a double-type Kalanchoë selection. A double-type hybrid progenyplant with desirable phenotypic characteristics is propagated asexuallyby conventional methods to determine if the phenotypic characteristicsare stable.

For example, a K. blossfeldiana (tetraploid)×K. laciniata (diploid)interspecific hybrid is by nature triploid and thus sterile. K.blossfeldiana times K. laciniata interspecific hybrid progeny plantswere screened and ‘Yellow African’, described in U.S. Plant Pat. No.12,299, was identified. This fertile K. blossfeldiana×K. laciniatainterspecific hybrid has been used to breed a series of interspecifichybrid cultivars designated African Treasures™. One such cultivar wasdesignated ‘KJ 2000 0716’ and is described in pending U.S. plant patentapplication Ser. No. 10/654,571.

‘KJ 2000 0716’ was identified in the progeny originating from a crossbetween ‘Yellow African’ and a single-type K. blossfeldiana. Three newdouble-flowered Kalanchoë interspecific hybrids originated from crossesbetween ‘KJ 2000 0716’ as the female parent, and ‘Monroe’ as maledouble-type K. blossfeldiana parent. ‘Monroe’ is described in U.S. PlantPat. No. 14,714.

Recurrent selection is used to increase the number of petals per flowerfound in the instant Kalanchoë interspecific hybrid plants. Adouble-type Kalanchoë interspecific hybrid plant is selfed, or crossedto another double-type Kalanchoë plant, and the progeny screened forplants with double-type flowers with an increased number of petals perflower compared to the double-type parents.

Similar methods as employed for double-type Kalanchoë selection are usedfor recurrent selection to optimize compactness found in the instantrol-transformed Kalanchoë interspecific hybrid plants. A rol-transformedKalanchoë interspecific hybrid plant is selfed, or crossed to anotherKalanchoë plant, and the progeny screened for compactness compared tothe rol-transformed parent.

D. Transformation Methodology: Transfer of Rol Genes

As explained above, transformation” refers to any methodology forintroducing a rol gene(s) into a host plant or plant cell. Importantly,and because A. rhizogenes naturally infects plants, transformationembraces transferring wild-type rol genes from wild-type bacterium intoa plant cell. Thus, and as used herein, transformation does not requirecloning a heterologous gene into a vector for transfer into a host plantcell, nor does transformation require genetically engineering thebacterium.

“Transformed plant” refers to a plant that comprises a nucleic acidsequence that also is present per se in another organism or species, orthat is optimized, relative to host codon usage, from another organismor species. Both monocotyledonous and dicotyledonous angiosperm orgymnosperm plant cells may be transformed in various ways known to theart. For example, see Klein et al., Biotechnology 4: 583-590 (1993);Bechtold et al., C. R. Acad. Sci. Paris 316: 1194-1199 (1993); Bent etal., Mol. Gen. Genet. 204: 383-396 (1986); Paszowski et al., EMBO J. 3:2717-2722 (1984); Sagi et al., Plant Cell Rep. 13: 262-266 (1994).Agrobacterium species such as A. tumefaciens and A. rhizogenes can beused, for example, in accordance with Nagel et al., Microbiol Lett 67:325 (1990). Additionally, plants may be transformed by Rhizobium,Sinorhizobium or Mesorhizobium transformation. Broothaerts et al.,Nature 433: 629-633 (2005).

For example, Agrobacterium may be transformed with a plant expressionvector via, e.g., electroporation, after which the Agrobacterium isintroduced to plant cells via, e.g., the well known leaf-disk method.Additional methods for accomplishing this include, but are not limitedto, electroporation, particle gun bombardment, calcium phosphateprecipitation, and polyethylene glycol fusion, transfer into germinatingpollen grains, direct transformation, Lorz et al., Mol. Genet. 199:179-182 (1985), and other methods known to the art. If a selectionmarker, such as kanamycin resistance, is employed, it makes it easier todetermine which cells have been successfully transformed. Marker genesmay be included within pairs of recombination sites recognized byspecific recombinases such as cre or flp to facilitate removal of themarker after selection. See U.S. published application No. 2004/0143874.

Transgenic plants without marker genes may be produced using a secondplasmid comprising a nucleic acid encoding the marker, distinct from afirst plasmid that comprises a rol sequence. The first and secondplasmids or portions thereof are introduced into the same plant cell,such that the selectable marker gene that is transiently expressed,transformed plant cells are identified, and transformed plants areobtained in which the rol sequence is stably integrated into the genomeand the selectable marker gene is not stably integrated. See U.S.published application No. 2003/0221213.

The Agrobacterium transformation methods discussed above are known fortransforming dicots. Additionally, de la Pena et al., Nature 325:274-276 (1987), Rhodes et al., Science 240: 204-207 (1988), andShimamato et al., Nature 328: 274-276 (1989) have transformed cerealmonocots using Agrobacterium. Also see Bechtold et al., C.R. Acad. Sci.Paris 316 (1994), illustrating vacuum infiltration forAgrobacterium-mediated transformation.

Plant cells may be transformed with a nucleic acid or nucleic acidconstruct without the use of a selectable or visible marker, andtransgenic organisms may be identified by detecting the presence of theintroduced sequence or construct. The presence of a protein,polypeptide, or nucleic acid molecule in a particular cell can bemeasured to determine if, for example, a cell has been successfullytransformed or transfected. For example, and as routine in the art, thepresence of the introduced construct can be detected by PCR or othersuitable methods for detecting a specific nucleic acid or polypeptidesequence. Additionally, transformed cells may be identified byrecognizing differences in the growth rate or a morphological feature ofa transformed cell compared to the growth rate or a morphologicalfeature of a non-transformed cell that is cultured under similarconditions. See WO 2004/076625.

Methods of regenerating a plant from a transformed cell or culture varyaccording to the plant species but are based on known methodology. Forexample, methods for regenerating Kalanchoë plants are well-known in theart can be found in Christensen, B., Sriskandarajah, S., Serek, M.,Müller, R., 2008. Transformation of Kalanchoë blossfeldiana withrol-genes is useful in molecular breeding towards compact growth. PlantCell Rep. 27, 1485-1495.

E. Plant Growth Conditions

The instant Kalanchoë plants described herein were grown in a greenhouseat 64.4 degree F. during the day and 68 degree F. during the night. Theplants were produced in pots with a diameter of 10.5 cm or 13 cm.Cuttings were grown under long-day conditions (16 hours light, 8 hoursnight) during the first 3-8 weeks following planting, depending oncultivar and pot size. Between 4-9 weeks after planting, the plants weretransferred to short-day conditions (10 hour light and 14 hour dark).The flowering is induced by short-day conditions. Between 13-19 weeksafter planting, depending on cultivar, pot size, and time of year, theplants were mature with flowers that were opening or about to open.

The plants were grown under natural light conditions supplemented with70.μmol photons m⁻² s⁻¹ SON-T light when the natural light was less than100.mu.mol/m2/s. Plants were grown in a peat based soil mix and werewatered with a solution containing 200 parts per million (ppm) nitrogen,200 ppm potassium, 40 ppm phosphorous, 200 ppm calcium, 40 ppmmagnesium, 60 ppm sulphate, 1 ppm iron, 0.6 ppm manganese, 0.1 ppmcopper, 0.1 ppm zinc, 0.3 ppm borium, 0.03 ppm molybdenum.

F. Selection and Analysis of Rol-Transformed Plants

The present rol-transformed plants are selected that contain and expressone or more rol genes relative to a control, non-transformed plant ofthe same species. Additionally, the instant plants may have an alteredphenotype relative to a non-transformed control plant. Such phenotypemay include an intermediate height or intermediate compactness, whereinthe transformed plant has a reduced height and/or compactness relativeto the control plant.

The phrase “intermediate height” refers to a quantitative reduction ofplant height relative to a wild-type or control plant of the samespecies. The height of the transformed plant can be decreased from about5% to about 60%, preferably from 10% to about 50%, even more preferablyfrom 15% to about 50% of the height of a wild type plant.

The phrase “intermediate compactness” refers to a quantitative reductionof plant compactness relative to a wild-type or control plant of thesame species. The compactness of the transformed plant can be increasedfrom about 5% to about 50%, preferably from 10% to about 40%, even morepreferably from 15% to about 50% of the height of a wild type plant.

The following examples are illustrative and do not limit the presentapplication. Of course, it is understood that many variations andmodifications can be made while remaining within the intended spirit andscope.

EXAMPLE 1 Transformation Materials and Methodology

Plant Material

In vivo plants of Kalanchoë grandiflora and the F1-hybrid 2009-0347(referred to as 0347) and established in vitro culture plants of K.blossfeldiana ‘Molly’, and K. grandiflora, and the F1-hybrid 2006-0199(referred to as 0199), (Knud Jepsen A/S, Hinnerup, Denmark and KU-LIFE,Crop Sciences, T∪strup). In vivo plants were cultivated in a greenhousewith temperatures of 20° C. at day and night, 16 hour day length and alight intensity of 260 μmol photons m⁻² s⁻¹. In vitro plants werecultivated in growth chamber with temperatures of 25° C. at day and 22°C. at night, 13 hour day length and a light intensity of 75 μmol photonsm⁻²s⁻¹. The two F1-hybrids are closely related since 0199 is thepaternal part of the crossing to produce 0347.

36, 104, 166, 158 and 128 leaf explants were used for transformation ofK. blossfeldiana ‘Molly’, 2006-0199, 2009-0347, K. grandiflora and K.grandiflora, respectively. 25 leaf explants for each species/hybrid wereused for control experiment. Leaves derived from in vivo material weresterilised in 70% EtOH for 1 min. followed by 20 min. in 1% NaOCl (VWR,Copenhagen, Denmark) and 0.03% (v/v) Tween 20 (Merck, La Jolla, USA) andwashed 3 times in sterile water and were stored until excision.

Bacterial Strain

Agrobacterium rhizogenes strain ATCC43057 (A4) (kindly provided by Dr.David Tepfer, Biologie de la Rhizosphère, INRA, Versailles, France) wasused for induction of hairy roots. The strain was cultured in liquid MYAmedium (Tepfer and Cassedelbart (1987) Microbiol Sci. 4, pp. 24-28. 1 mLof the bacterial glycerol stock (kept at −80° C.) was diluted in 10 mLMYA in a 50 mL Falcon tube and incubated for 8 h at 27° C. and shaken at260 rpm. The solution was further diluted with 100 mL MYA in a 250 mLflask and shaken at 260 rpm for 24 h in darkness at 27° C. TheOD₆₀₀=0.4-0.6 was measured on Nanodrop 1000 (Thermo Scientific,Wilmington, Del., USA).

Transformation

Sterilized leaves or in vitro plant were excised to pieces of min 1 cm×1cm and stored in sterile water until all explants were ready. The waterwas discarded from the explants and A. rhizogenes-suspension was addedto cover all explants for 30 min. After 30 min. the A.rhizogenes-suspension was discarded and the slices were transferred,with a thin layer of the A. rhizogenes suspension on the surface, toco-cultivation plates for 24 h in darkness without selection. Theexplants were cultivated in the lab at temperatures at 22° C. indarkness. After co-cultivation the explants were transferred to O-media(selection media) by drying the explants with pieces of ripped sterilefilter paper. The leaf surface was as dry as possible on both sides ofthe excised leaf. The explants stayed dark until roots were developedenough to be transferred to regeneration media. The material wastransformed over three sessions. Firstly transformation was conductedwith K. blossfeldiana ‘Molly’ and K. gracilipes, secondly; 0347 and K.grandiflora and finally with 0199. For each species/hybrid the controlsand putative transformants was performed the same day.

Basic Medium

The basic medium used as background of all media used was ½×MS (SigmaM0404) (consisting of Murashige and Skoog macro- and microelements)(Murashige and Skoog, 1962) at a concentration of 2.2 g L⁻¹, 30 g L⁻¹sucrose (table sugar), 7 g L⁻¹ bacto agar and 0.50 g L⁻¹2-(N-morpholino)-ethanesulphonic acid (MES) (Duchefa). The pH wasadjusted to 6.3 by 1 M KOH and the media was autoclaved at 121° C. and103.5 kPa.

Co-Cultivation Medium

Co-cultivation medium used for co-cultivation between explant and A.rhizogenes consisted of basic medium with 30 μg mL⁻¹ acetosyringone(Sigma-Aldrich, Steinheim, Germany).

Selection Medium

Selection medium was a hormone-free medium used for root formation ofputatively transformed explants and controls. Filter-sterilizedantibiotics were added after autoclaving to the selection media to thebasic medium. Selection media consist of basic media ½×MS medium withtimentin (TIM) in the concentration of 100 mg L⁻¹. Preferably, theselection medium contains arginine, preferably 0.5 mM arginine.

Regeneration Media

Regeneration medium containing the hormoneN-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) was used for regeneration ofnodules on the putatively transformed root clusters. Filter-sterilisedhormones and antibiotics were added after autoclaving to theregeneration media. The CPPU-medium contained basic ½×MS medium with 1.5μg L⁻¹ CPPU together with TIM in the concentration 100 mg L⁻¹.

Co-Cultivation

In all treatments the explants were co-cultivated for 24 hours. Afterco-cultivation, the explants were blotted onto sterile filter paper andthoroughly dried with ripped pieces of sterilised filter paper. Controlsand putatively transformed explants were transferred to selectionmedium.

Plant Selection

After 24 hours of co-cultivation the explants were transferred toO-media (selection medium) with 8 explants on each Petri dish, with anumber total number of Petri dishes of 5, 13, 21, 20 and 16 for ‘Molly’,2006-0199, 2009-0347, K. grandiflora and K. gracilipes, respectively.The increasing number of roots and decreasing number of explants (due tovitrification—the leaf sections became glass like or because ofinfections) were monitored for the specific Petri dish in the treatment.

Plant Regeneration

When the roots of putatively transformed explants had developed to alength of 1.5-2 cm they were transferred in clusters, with a part of theexplant to CPPU-medium. The transferred root clusters were placed in aclimate chamber (Celltherm, United Kingdom) on shelves with 11 hdaylight and day/night temperatures of 20/18° C. and an intensity of45-70 mmol photons M⁻²s⁻¹ (Philips, Amsterdam, The Netherlands). Onlyroot clusters with A. rhizogenes treated explants was transferred. Herethe number of root clusters was monitored as well as the number ofnodules developing from the roots. Counting of nodule development wasstopped when no positive development was observed after 30 days for anyof the five species/hybrids. Nodules from K. gracilipes were observedlosing colour and vigour. An attempt to stop this negative developmentwas made by the addition of 0.1 μg/ml Auxin (NAA). Result is stillunknown.

Control Plants

Control plants were treated like transformants but inoculated in MYAmedium without bacteria and with a lower number of explants-25 percultivar. The control experiment plants were conducted in parallel withthe transformants.

Molecular Analysis

DNA was isolated with DNeasy Plant Mini Kit (Qiagen, Hilden, Germany)from root clusters (and nodules) on regeneration medium. Half of a rootcluster was harvested for DNA extraction from each of the fivespecies/hybrids. The concentration was measured on NanoDrop 1000 (ThermoScientific, Wilmington, Del., USA). Concentrations was measured to 5.10ng/μl, 2.34 ng/μl, 4.34 ng/μl, 0.52 ng/μl and 2.06 ng/μl for 0199, 0347,K. grandiflora, K. gracilipes and K. blossfeldiana ‘Molly’,respectively. Since the concentration for K. gracilipes was too low thiswas not used for the PCR. PCR on DNA was preformed with a concentrationof 15 ng with the three specific primers (see Table 1 below) to amplifythe rolB gene on the T_(L)-DNA and KdActin and VirD2 as controls.Dimethyl sulfoxide (DMSO) was added to the PCR reaction to obtain abetter unfolding of the DNA. The following temperature program wasapplied for amplification in the DNA thermal cycler (Mycycler, Biorad,Hercules, Calif., USA): 95° C. for 10 min. (initial denaturation)followed by 40 cycles 95° C. for 30 sec. (denaturation), 58° C. for 30sec. (annealing) and 72° C. for 15 sec. (elongation), with a final 7min. elongation at 72° C. The amplified fragments sequences were mixedwith orange G (Sigma-Aldrich, SteinHeim, Germany) (40% sucrose (w/v) and1.5% Orange G (w/v) and Gelred (Biotium, Hayward, Calif., USA) andfractionated in 1% TAE agarose gel.

TABLE 1 Primer Sequences   (Lütken et al.,EuphyticaDOI 10.1007/s10681-012-0701-5.) Product Gene Primer sequence size (bp)rolA 5′-CCAATCTGAGCACCACTCCT-3′ 153 (SEQ ID NO: 9)5′-AATCCCGTAGGTTTGTTTCG-3′ (SEQ ID NO: 10) rolB5′-GATATCCCGAGGGCATTTTT-3′ 182 (SEQ ID NO: 11)5′-GAATGCTTCATCGCCATTTT-3′ (SEQ ID NO: 12) rolC5′-CAATAGAGGGCTCAGGCAAG-3′ 202 (SEQ ID NO: 13)5′-CCTCACCAACTCACCAGGTT-3′ (SEQ ID NO: 14) rolD5′-GCGAAGTGGATGTCTTTGG-3′ 225 (SEQ ID NO: 15)5′-TTGCGAGGTACACTGGACTGA-3′ (SEQ ID NO: 16) KdActin*5′-GCAGGACGTGATCTGACTGA-3′ 168 (SEQ ID NO: 17)5′-GACGGACGAGCTACTCTTGG-3′ (SEQ ID NO: 18)Statistical Analysis

K. blossfeldiana ‘Molly’, 0199, 0347, K. grandiflora and K. grandiflora,had a total number of petri dishes of 5, 13, 21, 20 and 16, and a totalnumber of explants of 36, 104, 166, 158, respectively, K. blossfeldiana‘Molly’ functioned as a reference of the transformation. Similarly,control explants had five replicates but 5 explants per species/hybridswith a total of 25 per species/hybrids. Since the explants may be takenout of the experiment because of infection, the number of explantschanged over time. The total number of explants was therefore monitoredto obtain a better ratio between number of explants and formation ofroots. The number of roots was monitored as the number increased. Theaverage of surviving explants per petri dish and the average of rootsper petri dish were calculated. The two averages were used to calculatea ratio for each petri dish to describe the number of roots perexplants.

V_(max) (root development/days) was modelled with a linear regressionand using the slope. The calculations were performed in Excel. Standarddeviations (SD) and students t-test (t-test) were calculated for eachobservation to verify variation within the individual species/hybrids.SD and t-test was calculated in Excel. ANOVA test was performed with R.

Results

The experiments involved a natural transformation with Agrobacteriumrhizogenes to study the transformation efficiency for different speciesand hybrids and for plain material from in vivo and in vitro. Twospecies; K. gracilipes and K. grandiflora and two hybrids; 2006-0199 and2009-0347 was transformed with the conditions that was found optimal forK. blossfeldiana ‘Molly’ by Christensen et al., (2008). K. blossfeldiana‘Molly’ was used as a control within the transformants since thecultivar formed background of the transformation system.

Root induction and growth were monitored as a total number of roots perpetri dish in each treatment. Since some explants were removed due toinfection the total number of explants over time was also monitored.This was done to obtain a more unbiased assessment when calculating thenumber of roots per explant in each plant line.

Root Development on 0-Media

Roots from K. blossfeldiana ‘Molly’ and K. gracilipes had a later timeto first transfer to regeneration medium (88 and 77 days, respectively)compared to 0199, 0347 and K. grandiflora (45, 38 and 38 days,respectively). At the time of the first time of transfer to regenerationmedium putative transformants from 0199, K. grandiflora, K. gracilipesand K. blossfeldiana ‘Molly’ were significantly different from control.Only 0347 was not significantly different at the time of first transferof root clusters, though data show that this changed after 45 days onselection medium (data not shown).

K. gracilipes developed roots fastest (16 days from transfer fromco-cultivation medium to selection medium) followed by K. grandifloraand 0199 (both 17 days after transfer) and K. blossfeldiana ‘Molly’ (18days after transfer) and finally 0347 (24 days after transfer).

At the first day of transfer to regeneration medium, K. blossfeldiana‘Molly’ had the highest number of roots per explant (19.4 roots perexplant in average), hereafter 0199 (8.5 roots per explant in average)and K. grandiflora (3.1 roots per explant in average) and finally 0347and K. gracilipes (both 2.4 roots per explant in average).

What is claimed is:
 1. A species-independent method for transforming aKalanchoë interspecific hybrid plant, comprising: (a) co-cultivatingwild-type A. rhizogenes with a Kalanchoë interspecific hybrid plant,wherein A. rhizogenes transfers one or more rol genes into said plant,wherein said one or more rol genes are selected from the groupconsisting of rolA, rolB, rolC, and rolD, wherein said one or more rolgenes at least includes rolB, wherein the nucleic acid sequence forrolA, rolB, rolC, and rolD has 100% sequence identity with SEQ ID NOs:3, 4, 5, and 6, respectively; (b) selecting a putatively transformedroot having a hairy root phenotype; (c) growing said root on aregeneration medium; (d) regenerating a shoot from said root, therebygenerating a plantlet, and; (e) growing said plantlet into a matureplant.
 2. The method of claim 1, further comprising assaying thepresence of one or more rol genes in said mature plant.
 3. A method forreducing the height of a Kalanchoë interspecific hybrid plant by about5% to about 60%, compared to a wild-type control plant, comprising: (a)transforming Kalanchoë plant tissue with A. rhizogenes, wherein A.rhizogenes delivers and integrates one or more rol genes into hybridplant genome, wherein said one or more rol genes are selected from thegroup consisting of rolA, rolB, rolC, and rolD, wherein said one or morerol genes at least includes rolB, wherein the nucleic acid sequence forrolA, rolB, rolC, and rolD has 100% sequence identity with SEQ ID NOs:3, 4, 5, and 6, respectively; (b) selecting a putatively transformedroot having a hairy root phenotype; (c) growing said root on aregeneration medium; (d) regenerating a shoot from said root, therebygenerating a plantlet; and (e) growing said plantlet into a matureplant, and; (f) selecting a plant having a reduced height by about 5% toabout 60% compared to the height of a non-transformed control plant ofthe same species.
 4. A rol-transformed Kalanchoë interspecific hybridwith intermediate height, wherein said intermediate height is areduction of height by about 5% to about 60% of the height of a control,non-transformed Kalanchoë interspecific hybrid plant, wherein therot-transformed Kalanchoë interspecific hybrid has been transformed withone or more rol genes are selected from the group consisting of rolA,rolB, rolC, and rolD, wherein said one or more rol genes at leastincludes rolB, wherein the nucleic acid sequence for rolA, rolB, rolC,and rolD has 100% sequence identity with SEQ ID NOs: 3, 4, 5, and 6,respectively.
 5. Progeny of a rol transformed Kalanchoë interspecifichybrid of claim 4.